Endotoxin is a lipopolysaccharide which is present in the cell wall outer membrane of Gram-negative bacteria and is known as a strong pyrogen. Endotoxin is also known to cause various pathological conditions including not only fever but also shock and the like. Therefore, detection and quantitation of endotoxin is essential in pharmaceutical products such as an injection pharmaceutical preparation, water, medical devices, and the like, from the viewpoint of ensuring safety of pharmaceutical products and the like.
As has been known, a horseshoe crab undergoes blood clotting when it is infected with a Gram-negative bacterium. This phenomenon has been conventionally employed for the detection of endotoxin. That is, endotoxin can be determined by use of blood corpuscle extracts of horseshoe crab (i.e., limulus amebocyte lysate, hereinafter referred to as “LAL”). This method is called “limulus test,” which employs a cascade reaction of a variety of proteins (all serine proteases) present in LAL, which reaction occurs via contact between endotoxin with LAL.
AT III is an anti-coagulant in blood and is used for the treatment of disseminated intravascular coagulation (DIC) or the like. Since AT III is a serine protease inhibitor, it inhibits the activities of proteins which are present in LAL and which are involved in the cascade reaction (Non-Patent Document 1). Thus, in some cases, a certain type of a specimen inhibits a limulus reagent. Such an inhibitory action may be avoided by diluting a specimen (Non-Patent Document 2). However, AT III exhibits a very strong inhibitory action on a limulus reagent, and therefore, in order to carry out limulus test of an AT III pharmaceutical preparation (injection), the preparation must be diluted at a dilution factor of 64 or higher (Non-Patent Document 2). Therefore, in consideration of the detection sensitivity of a limulus reagent (i.e. the lowest endotoxin concentration on the calibration curve), subjecting an AT III pharmaceutical preparation to limulus test has been considered to be difficult (Non-Patent Document 2). Actually, even at present, detection of endotoxin in specimens each containing AT III is performed through the rabbit pyrogen test (Non-Patent Document 1).
Non-Patent Document 1 discloses a method for determining Et in an AT III pharmaceutical preparation, in which method the AT III pharmaceutical preparation is 100-fold diluted; the diluted product is heated at 70° C. for 20 minutes as a pre-treatment; the pre-treated product is reacted with LAL; and Et is detected through the light scattering method. A technical feature of the method resides in employment of the light scattering method by means of a light scattering measurement apparatus instead of turbidimetric assay by means of a spectrophotometer, in order to realize a high sensitivity in Et detection. However, similar to the aforementioned case, the method requires dilution of a specimen at high dilution factor, and involves at least the following problems.
(1) Spectrophotometers are widely employed as a measurement apparatus for limulus test, and a light scattering measurement apparatus must be additionally provided and used for carrying out the light scattering method.
(2) The Japanese Pharmacopoeia specifies only turbidimetric assay and chromogenic assay as photometric assay methods of Et, and is silent to a light scattering method.
Patent Document 1 discloses an Et and BG assay method for biological samples, the method comprising bringing a biological sample into contact with an alkaline earth metal sulfate or an alkaline earth metal halide, heating the mixture as a pre-treatment, and then subjecting the pre-treated product to an assay by use of a limulus reagent.
Patent Document 2 discloses an Et and BG assay method for blood samples, the method comprising mixing a biological sample with a hexadimethrine compound, an alkali metal hydroxide, a nonionic surfactant, and an alkaline earth metal halide, heating the mixture as a pre-treatment, and then subjecting the pre-treated product to an assay by use of a limulus reagent.
Patent Document 3 discloses an Et assay method for a sample possibly containing AT III, the method comprising bringing a biological sample into contact with a carrier on which lipopolysaccharide-binding peptide and/or lipid A-binding peptide is immobilized to thereby recover Et via absorption into the carrier, and then subjecting the recovered Et to an assay by use of a limulus reagent or the like.
However, none of the aforementioned documents discloses or suggests subjecting AT III to a protein inactivation treatment as a pre-treatment in the co-presence of a divalent metal salt for carrying out limulus test of AT III.